One of the primary goals of plant genetic research is to provide transgenic plants which express a foreign gene in an amount sufficient to confer the desired phenotype to the plant. Significant advances have been made in pursuit of this goal, but the expression of some foreign genes in transgenic plants remains problematic. It is believed that numerous factors are involved in determining the ultimate level of expression of a foreign gene in a plant, and the level of mRNA produced in the plant cells is believed to be a major factor that limits the amount of a foreign protein that is expressed in a plant.
It has been suggested that the low levels of expression observed for some foreign proteins expressed in monocotyledonous plants (monocots) may be due to low steady state levels of mRNA in the plant as a result of the nature of the coding sequence of the structural gene. This could be the result of a low frequency of full-length RNA synthesis caused by the premature termination of RNA during transcription or due to unexpected MRNA processing during transcription. Alternatively, full-length RNA could be produced, but then processed by splicing or polyA addition in the nucleus in a fashion that creates a nonfunctional mRNA. It is also possible or the MRNA to be properly synthesized in the nucleus, yet not be suitable for sufficient or efficient translation in the plant cytoplasm.
Various nucleotide sequences affect the expression levels of a foreign DNA sequence introduced into a plant. These include the promoter sequence, intron sequences, the structural coding sequence that encodes the desired foreign protein, 3' untranslated sequences, and polyadenylation sites. Because the structural coding region introduced into the plant is often the only "non-plant" or "non-plant related" sequence introduced, it has been suggested that it could be a significant factor affecting the level of expression of the protein. In this regard, investigators have determined that typical plant structural coding sequences preferentially utilize certain codons to encode certain amino acids in a different frequency than the frequency of usage appearing in bacterial or non-plant coding sequences. Thus it has been suggested that the differences between the typical codon usage present in plant coding sequences as compared to the typical codon usage present in the foreign coding sequence is a factor contributing to the low levels of the foreign mRNA and foreign protein produced in transgenic monocot plants. These differences could contribute to the low levels of MRNA or protein of the foreign coding sequence in a transgenic plant by affecting the transcription or translation of the coding sequence or proper mRNA processing. Recently, attempts have been made to alter the structural coding sequence of a desired polypeptide or protein in an effort to enhance its expression in the plant. In particular, investigators have altered the codon usage of foreign coding sequences in an attempt to enhance its expression in a plant. Most notably, the sequence encoding insecticidal crystal proteins of B. thuringiensis (B.t.) has been modified in various ways to enhance its expression in a plant, particularly monocotyledonous plants, to produce commercially viable insect-tolerant plants.
In the European Patent Application No. 0359472 of Adang et al., a synthetic B.t. toxin gene was suggested which utilized codons preferred in highly expressed monocotyledonous or dicotyledonous proteins. In the Adang et al. gene design, the resulting synthetic gene closely resembles a typical plant gene. That is, the native codon usage in the B.t. toxin gene was altered such that the frequency of usage of the individual codons was made to be nearly identical to the frequency of usage of the respective codons in typical plant genes. Thus, the codon usage in a synthetic gene prepared by the Adang et al. design closely resembles the distribution frequency of codon usage found in highly expressed plant genes.
Another approach to altering the codon usage of a B.t. toxin gene to enhance its expression in plants was described in Fischhoffet al., European Patent Application No. 0385962. In Fischhoff et al., a synthetic plant gene was prepared by modfing the coding sequence to remove all ATTTA sequences and certain identified putative polyadenylation signals. Moreover, the gene sequence was preferably scanned to identify regions with greater than four consecutive adenine or thymine nucleotides and if there were more than one of the minor polyadenylation signals identified within ten nucleotides of each other, then the nucleotide sequence of this region was altered to remove these signals while maintaining the original encoded amino acid sequence. The overall G+C content was also adjusted to provide a final sequence having a G+C ratio of about 50%.
PCT Publication No WO 91/16432 of Cornelissen et al. discloses a method of modifying a DNA sequence encoding a B.t. crystal protein toxin wherein the gene was modified by reducing the A+T content by changing the adenine and thymine bases to cytosine and guanine while maintaining a coding sequence for the original protein toxin The modified gene was expressed in tobacco and potato. No data was provided for maize or any other monocot.